Expression and properties of two distinct classes of the phorbol ester receptor family, four conventional protein kinase C types, and a novel protein kinase C.
Five rabbit cDNAs, encoding four conventional protein kinase Cs (PKCs), alpha, beta I, beta II, and gamma, and a novel PKC-related protein (nPKC epsilon) were transfected into COS cells. Antisera raised against a bacterially synthesized fragment of PKC alpha or nPKC epsilon and against a chemically synthesized peptide of PKC beta I or beta II, specifically identified the corresponding species in the transfected cells. All four PKCs and nPKC epsilon expressed by transfection served as phorbol ester receptors. Phorbol 12,13-dibutyrate (PDBu)-binding activities of all PKCs and nPKC epsilon required phospholipid but not magnesium. The phosphatidylserine requirement for the activity of nPKC epsilon is independent of Ca2+ and similar to that for PKC alpha observed at 0.03 mM Ca2+. Calcium dependence of the binding activity was observed only for the four conventional PKCs. Scatchard plot analysis clearly showed that the dissociation constants of PDBu for all four PKCs were nearly the same (approximately 25 nM) in the presence of Ca2+, and that the value for nPKC epsilon was slightly higher (84 nM) and independent of Ca2+. The latter value is comparable to those observed in several cell types under conditions of Ca2+ chelation. Translocation of conventional PKC alpha to the membranes was induced with phorbol ester in a Ca2+-dependent manner, whereas the PDBu-stimulated translocation of nPKC epsilon did not require Ca2+. These results, together with previous studies on the enzymological characteristics of nPKC epsilon (Ohno, S., Akita, Y., Konno, Y., Imajoh, S., and Suzuki, K. (1988) Cell 53, 731-741), suggest that nPKC epsilon plays an important role in a transmembrane signaling pathway distinct from that involving conventional PKCs.
Akita
Y
Y
Department of Molecular Biology, Tokyo Metropolitan Institute of Medical Science, Japan.
Ohno
S
S
Konno
Y
Y
Yano
A
A
Suzuki
K
K
D000818:Animals; D015153:Blotting, Western; D029742:Caenorhabditis elegans Proteins; D002118:Calcium / Q000494:pharmacology; D002352:Carrier Proteins; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004247:DNA / Q000235:genetics; D015342:DNA Probes; D015870:Gene Expression; D009693:Nucleic Acid Hybridization; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism / Q000494:pharmacology; D010718:Phosphatidylserines / Q000494:pharmacology; D011493:Protein Kinase C / Q000235:genetics* / Q000378:metabolism; D011817:Rabbits; D011955:Receptors, Drug / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D015342:DNA Probes; D010718:Phosphatidylserines; D011955:Receptors, Drug; C083141:phorbol ester binding protein; C027776:phorbol ester receptor; D015240:Phorbol 12,13-Dibutyrate; D004247:DNA; D011493:Protein Kinase C; D002118:Calcium